Peptides acting as both GLP-1 receptor agonists and glucagon receptor antagonists and their pharmacological methods of use

ABSTRACT

The invention provides polypeptides that act both as an agonist of the GLP-1 receptor and an antagonist of the glucagon receptor. Such polypeptides are useful for treating individuals with type 2 diabetes or other metabolic disorders.

[0001] This application is a continuation-in-part of U.S. Ser. No.10/265,345, filed Oct. 3, 2002.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polypeptides that actboth as an agonist of the GLP-1 receptor and an antagonist of theglucagon receptor and the use of such polypeptides for therapeuticpurposes. More particularly, polypeptides of the present invention areuseful in stimulating the release of insulin from pancreatic beta cellsin a glucose-dependent manner and reducing glucagon-mediated secretionof glucose from the liver, thereby providing a treatment option forthose individuals afflicted with a metabolic disorder such as diabetes,hyperglycemia, impaired fasting glucose, impaired glucose tolerance,prediabetic states, and obesity.

BACKGROUND OF THE RELATED ART

[0003] Diabetes is characterized by impaired insulin secretionmanifesting itself among other things by an elevated blood glucose levelin the diabetic patient. Underlying defects lead to a classification ofdiabetes into two major groups: type I diabetes (or insulin dependentdiabetes mellitus, IDDM), which arises when patients lackinsulin-producing beta-cells in their pancreatic glands, and type 2diabetes (or non-insulin dependent diabetes mellitus, NIDDM), whichoccurs in patients with an impaired beta-cell insulin secretion and/oralterations in insulin action.

[0004] Type 1 diabetic patients are currently treated with insulin,while the majority of type 2 diabetic patients can be treated withagents that stimulate beta-cell function or with agents that enhance thetissue sensitivity of the patients towards insulin. Over time, almostone-half of type 2 diabetic subjects lose their response to these agentsand then must be placed on insulin therapy. The drugs presently used totreat type 2 diabetes include alpha-glucosidase inhibitors (PRECOSE®,VOGLIBOSE™, and MIGLITOL®), insulin sensitizers (e.g., Avandia™, Actos™and Rezulin™), insulin secretagogues (sulfonylureas (“SFUs”) and otheragents that act by the ATP-dependent K⁺ channel), and GLUCOPHAGE™(metformin HCl).

[0005] Alpha-glucosidase inhibitors. Alpha-glucosidase inhibitors reducethe excursion of postprandial glucose by delaying the absorption ofglucose from the gut. These drugs are safe and provide treatment formild to moderately affected diabetic subjects. However, gastrointestinalside effects have been reported in the literature and limit theireffectiveness.

[0006] Insulin sensitizers. Insulin sensitizers are drugs that enhancethe body's response to insulin. Thiozolidinediones such as Avandia™(rosiglitazone) and Actos™ activate peroxisome proliferator-activatedreceptor (PPAR) gamma and modulate the activity of a set of genes thathave not been well described. Hepatic effects (e.g., drug inducedhepatotoxicity and elevated liver enzyme levels) do not appear to be asignificant problem in patients using Avandia™ and Actos™. Even so,liver enzyme testing is recommended every two months in the first yearof therapy and periodically thereafter. Avandia™ and Actos™ treatmentsare associated with fluid retention, edema, and weight gain. Avandia™ isnot indicated for use with insulin because of concern about congestiveheart failure. Rezulin™ (troglitazone), the first drug in this class,was withdrawn because of elevated liver enzyme levels and drug-inducedhepatotoxicity.

[0007] Insulin secretagogues. Sulfonylureas (SFUs) and thenon-sulfonylureas, Nateglinide and Pepaglinide act through theATP-dependent potassium channel to cause glucose-independent insulinsecretion. These drugs are standard therapy for type 2 diabetics thathave mild to moderate fasting hyperglycemia. The insulin secretagogueshave limitations that include a potential for inducing hypoglycemia,weight gain, and high primary and secondary failure rates. Ten to 20% ofinitially treated patients fail to show a significant treatment effect(primary failure). Secondary failure is demonstrated by an additional20-30% loss of treatment effect after six months of treatment withinsulin secretagogues. Insulin treatment is required in 50% of theinsulin secretagogues responders after 5-7 years of therapy (Scheen etal., Diabetes Res. Clin. Pract. 6:533-543, 1989). Nateglinide andPepaglinide are short-acting drugs that need to be taken three times aday. They are used only for the control of post-prandial glucose and notfor control of fasting glucose.

[0008] GLUCOPHAGE™ is a biguanide that lowers blood glucose bydecreasing hepatic glucose output and increasing peripheral glucoseuptake and utilization. The drug is effective at lowering blood glucosein mildly and moderately affected subjects and does not have a sideeffect of weight gain or a potential to induce hypoglycemia. However,GLUCOPHAGE™ has a number of side effects, including gastrointestinaldisturbances and lactic acidosis. GLUCOPHAGE™ is contraindicated indiabetics over the age of 70 and in subjects with impaired renal orliver function. Finally, GLUCOPHAGE™ has the same primary and secondaryfailure rates as the insulin secretagogues.

[0009] Insulin treatment is instituted after diet, exercise, and oralmedications have failed to control blood glucose adequately. Thistreatment has several drawbacks: it is an injectable, it can producehypoglycemia, and it can cause weight gain. The possibility of inducinghypoglycemia with insulin limits the extent that hypoglycemia can becontrolled.

[0010] Problems with current treatments necessitate new therapies totreat type 2 diabetes. In particular, new treatments to retain normal(i.e., glucose-dependent) insulin secretion are needed. Givenglucagon-like peptide-1's (“GLP-1”) role in promoting glucose-regulatedinsulin secretion in the pancreas, GLP-1 receptor agonists arepotentially valuable in the treatment of such diseases. Moreover,glucagon receptor antagonists should prove valuable in treating type 2diabetes given glucagon's role in elevating plasma glucose bystimulating hepatic glycogenolysis and gluconeogenesis.

[0011] GLP-1 and glucagon are members of a family of structurallyrelated peptide hormones, the glucagon/secretin family. Within thisfamily, GLP-1(7-36) and GLP-1(7-37) (30 amino acids and 31 amino acids,respectively) and glucagon (30 amino acids) constitute a highlyhomologous set of peptides. In addition, these two hormones originatefrom a common precursor, preproglucagon which, upon tissue-specificprocessing, leads to production of GLP-1 predominantly in the intestineand glucagon in the pancreas. The receptors for these two peptides arehomologous (58% identity) and belong to the family of G-protein coupledreceptors.

[0012] GLP-1 and glucagon both play major roles in overall glucosehomeostasis. GLP-1 lowers plasma glucose concentrations mediated byglucose dependent insulin secretion, whereas glucagon increases plasmaglucose concentrations. Given the important roles of both GLP-1 andglucagon in maintaining normal blood glucose concentrations, there hasbeen considerable interest in the identification of GLP-1 receptoragonists and glucagon receptor antagonists. Clinical studies havedemonstrated the ability of GLP-1 infusion to promote insulin secretionand to normalize plasma glucose in diabetic subjects. However, GLP-1 israpidly degraded and has a very short half-life in the body.Furthermore, GLP-1 causes gut motility side effects at or near itstherapeutic doses. Therefore, GLP-1 itself has significant limitationsas a therapeutic agent, and modified versions of the peptide withenhanced stability are being pursued. Non-peptide agonists of the GLP-1receptor have not been described to date.

[0013] Peptide analogs of glucagon have been identified which act asglucagon antagonists and reduce hyperglycemia in diabetic rats. However,no peptide glucagon antagonist has, moved beyond preclinicaldevelopment. A number of structurally diverse non-peptide glucagonreceptor antagonists have been reported in the scientific and patentliterature. However, attempts to identify small molecule inhibitors ofthe glucagon receptor have met with limited success in vivo. The onlyantagonist of glucagon action known to be active in a clinical study isa compound identified as BAY 27-9955. A potential side effect ofglucagon antagonism is hypoglycemia.

[0014] Because of the potential side effects associated withadministering either a GLP-1 receptor agonist or a glucagon receptorantagonist alone, a combination therapy would have an advantage ofmaintaining the desired lowering of blood glucose while reducing theside effects. Co-administration, however, requires a single formulationand delivery approach that yields appropriate pharmokinetic profiles forboth peptides. This could be a major obstacle to the development of sucha therapeutic.

[0015] Based on the foregoing, considerable potential exists for asingle therapeutic peptide functioning as both a GLP-1 agonist and aglucagon antagonist in vivo.

SUMMARY OF THE INVENTION

[0016] This invention provides novel polypeptides that function both asan agonist of the GLP-1 receptor and an antagonist of the glucagonreceptor and which are effective in the treatment of diseases andconditions that can be ameliorated by agents having both GLP-1 receptoragonist and glucagon receptor antagonist activity. Polypeptides of thepresent invention provide a new therapy for patients with, for example,metabolic disorders, such as those resulting from decreased endogenousinsulin secretion, in particular type 2 diabetics, or for patients withimpaired glucose tolerance, a prediabetic state that has a mildalteration in insulin secretion or impaired fasting glucose, or obesity.

[0017] One aspect of the invention is a polypeptide selected from thegroup consisting of SEQ ID NOS: 6-32, as well as fragments, derivativesand variants of polypeptides that function as both an agonist of theGLP-1 receptor and an antagonist of the glucagon receptor atsubstantially the same level as the polypeptides shown in SEQ ID NOS:6-32 (collectively, “polypeptides of the invention”).

[0018] Other embodiments of the invention include polynucleotides thatencode polypeptides of the invention and the attendant vectors and hostcells necessary to recombinantly express the polypeptides.

[0019] Still other embodiments of the invention provide methods oftreating diabetes and/or other diseases or conditions affected bypolypeptides of the invention in mammals, including humans. The methodsinvolve administering a therapeutically effective amount of any of thepolypeptides of the present invention to a mammal.

[0020] The invention also provides recombinant and synthetic methods ofmaking polypeptides of the invention.

BRIEF DESCRIPTION OF THE DRAWING

[0021]FIG. 1 is a restriction map of a typical plasmid containing aGST-peptide fusion polynucleotide coding sequence.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The invention provides novel polypeptides, as well as fragments,derivatives variants, and analogs thereof that function as both anagonist of the GLP-1 receptor and an antagonist of the glucagonreceptor. Polypeptides of the invention function in vivo as both GLP-1receptor agonists and glucagon receptor antagonists in the preventionand/or treatment of such diseases or conditions as diabetes,hyperglycemia, impaired glucose intolerance, impaired fasting glucose,and obesity.

[0023] GLP-1 and glucagon are members of a family of structurallyrelated peptide hormones, the glucagon/secretin family. The stackingalignment below shows the primary structural relationships: GlucagonHSQGTFTSDYSKYLEGQAAKEFIAWLVKGR (SEQ ID NO:1) GLP-1 (7-36)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂ (SEQ ID NO:2) GLP-1 (7-37)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO:3)

[0024] Single-letter abbreviations for amino acids can be found inZubay, Biochemistry 2d ed., 1988, MacMillan Publishing, New York, p. 33.These polypeptides play a role in overall glucose homeostasis: GLP-1lowers plasma glucose concentrations, whereas glucagon increases plasmaglucose concentrations.

[0025] Given GLP-1's role in promoting glucose-regulated insulinsecretion in the pancreas, GLP-1 receptor agonists are potentiallyvaluable in the treatment of metabolic disorders and other diseases.Moreover, glucagon receptor antagonists should also prove valuable intreating disease given glucagon's role in elevating plasma glucose bystimulating hepatic glycogenolysis and gluconeogenesis. However, thesefacts alone do not guarantee glucose reduction in vivo without inducingsignificant side effects.

[0026] The invention provides new polypeptides that are both GLP-1receptor agonists and glucagon receptor antagonists. Without being boundto theory, we believe that polypeptides of the invention are capable ofreducing glucose levels in vivo by stimulating insulin release frompancreatic beta cells in a glucose-dependent manner, while at the sametime reducing glucagon-mediated secretion of glucose from the liver.

[0027] GLP-1 Receptor Agonist and Glucagon Receptor AntagonistPolypeptides

[0028] Polypeptides of the invention function both as a GLP-1 receptoragonist and a glucagon receptor antagonist. The GLP-1 receptor agonistcomponent of such polypeptides activates the GLP-1 receptor in one ormore in vitro or in vivo assays for GLP-1 receptor activation. Examplesof such assays include, but are not limited to, in vitro assays forinduction of cAMP in RINm5F cells, in vitro assays for induction ofinsulin secretion from pancreatic β-cells, in vivo assays for reductionin plasma glucose levels, and in vivo assays for elevation in plasmainsulin levels as described in the specific examples below.

[0029] Polypeptides of the invention also demonstrate glucagon receptorantagonist activity in one or more in vitro or in vivo assays forinhibition of glucagon receptor activation. Examples of such assaysinclude, but are not limited to, in vitro assays to measure inhibitionof glucagon-mediated increase in cellular cAMP, in vitro assays tomeasure inhibition of glucagon-mediated glucose release from, forexample, cultured hepatocytes, or in vivo assays for glucagon stimulatedglucose production as described in the specific examples below.

[0030] Preferred polypeptides of the invention are selected from thegroup consisting of (1) SEQ ID NOS: 6-32 and (2) fragments, derivatives,variants, and analogs thereof that function as both an agonist of theGLP-1 receptor and an antagonist of the glucagon receptor atsubstantially the same level as any of the polypeptides shown in SEQ IDNOS: 6-32.

[0031] Polypeptides of the present invention may be naturally-occurringpolypeptides, recombinant polypeptides, or synthetic polypeptides.

[0032] Fragments, Derivatives, Variants, and Analogs

[0033] Fragment, derivative, variant, and analog polypeptides retainsubstantially the same biological function or activity as, for example,polypeptides shown in SEQ ID NOS: 6-32. “Substantially the samebiological function or activity” is about 30% to 100% (i.e., 30, 40, 50,60, 70, 80, 90, or 100%) or more of the same biological activity of thefull-length polypeptide to which it is compared.

[0034] Derivatives

[0035] Derivatives include polypeptides of the invention that have beenchemically modified to provide an additional structure and/or function.For example, polyethylene glycol (PEG) or a fatty acid can be added to apolypeptide to improve its half-life. Fusion polypeptides which confertargeting specificity or an additional activity also can be constructed,as described in more detail below.

[0036] Derivatives can be modified by either a natural process, such asposttranslational processing, or by chemical modification techniques,both of which are well known in the art. Modifications can occuranywhere in a polypeptide, including the peptide backbone, the aminoacid side-chains, and the amino or carboxyl termini. It will beappreciated that the same type of modification may be present to thesame or varying degrees at several sites in a given polypeptide. Also, avariant may contain one or more different types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.

[0037] Other chemical modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formulation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, T. E. Creighton, PROTEINS, STRUCTURE AND MOLECULARPROPERTIES, 2nd ed., W. H. Freeman and Company, New York (1993);POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, ed.,Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.Enzymol 182:626-46 (1990); Rattan et al., Ann. N.Y. Acad. Sci.663:48-62, 1992).

[0038] Derivatives also include mature polypeptides that have been fusedwith another polypeptide, such as for example human serum albumin, toimprove their pharmacokinetic profile. Fusion of two polypeptides can beaccomplished by any means known to one skilled in the art. For example,a DNA encoding human serum albumin and a DNA sequence encoding apolypeptide of the invention can be cloned into any mammalian expressionvector known to one skilled in the art. Location of a polypeptide of theinvention N-terminal to the other polypeptide is preferred, because itappears that a free N-terminal histidine is required for GLP-1 receptoractivity (Kawa, Endocrinology Apr;124(49):1768-73, 1989). The resultingrecombinant fusion protein can then be expressed by transforming asuitable cell line, such as HKB or CHO, with the vector and expressingthe fusion protein.

[0039] Preferred derivatives include polypeptides of the invention (SEQID NOS: 6-32) to which a PEG moiety or a fatty acid moiety has beenattached. The PEG moiety can be, for example, a PEG with a molecularweight greater than 22 kDa, preferably a molecular weight of between 25kDa and 100 kDa (e.g., 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 kDa), and more preferably a molecular weight of between 35kDa and 45 kDa (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45kDa). Examples of such PEGylated polypeptides are those that contain a22 kDa PEG moiety attached to the cysteine residue at position 31 of SEQID NOS: 19 or 25. See SEQ ID NOS: 20 and 26 and Table 2. Alternatively,a 43 kDa PEG moiety can be attached to the cysteine residue at position31 of SEQ ID NO: 25. See SEQ ID NO: 27 and Table 2. A PEG moiety can beadded to a cysteine residue of polypeptides of the invention by methodswell known in the art, for example, see Example 19.

[0040] Other preferred derivatives have a fatty acid moiety attached tothe polypeptide. The fatty acid moiety can be, for example, a fatty acidbetween C₁₂ and C₂₀, preferably C₁₄ and C₁₈, and most preferably a C₁₆fatty acid. Examples of such polypeptides are SEQ ID NOS: 28-32, whichcontain a C₁₆ (palmitate) fatty acid moiety attached to a Tysine residuewithin the peptide. See Table 2. A fatty acid moiety can be added to alysine residue of polypeptides of the invention by methods well known inthe art, for example, fatty acid acylation (Knudsen et al., J. Med.Chem. 43:1664-1669, 2000).

[0041] Variants

[0042] Variants are polypeptides on the invention that have or moreamino acid sequence changes with respect to the amino acid sequencesshown in SEQ ID NOS: 6-32. Variants also can have amino acids joined toeach other by modified peptide bonds, i.e., peptide isosteres, and maycontain amino acids other than the 20 naturally occurring amino acids.

[0043] Preferably, variants contain one or more conservative amino acidsubstitutions (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions),preferably at nonessential amino acid residues. A “nonessential” aminoacid residue is a residue that can be altered from a wild-type sequenceof a protein without altering its biological activity, whereas an“essential” amino acid residue is required for biological activity. Aconservative amino acid substitution is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Non-conservativesubstitutions would not be made for conserved amino acid residues or foramino acid residues residing within a conserved protein domain.

[0044] Conservative amino acid substitutions are preferably at positions11, 12, 16, 17, or 18 of the consensus polypeptide shown SEQ ID NO: 34.Position 11 preferably is R, S, A, K, G, or T and more preferably R, A,G, or S. Position 12 preferably is K, N, R, H, A, S or Q and morepreferably K, A, S, or N. Position 16 preferably is K, R, V, I, L, M, F,W, Y, A, S, T, N, Q, G, or H, and more preferably K, V, I, F, A, S, orN. Position 17 preferably is D, E, H, K, R, F, I, L, M, Y, V, W, A, S,T, N, Q, or G, and more preferably R, A, L, M, V, S, H, E, or Q.Position 18 preferably is K, R, F, I, L, Y, V, M, A, G, or H and morepreferably K, R, F, I, L, Y or A. All possible combinations ofsubstitutions at positions 11, 12, 16, 17, and 18, including nosubstitution at any one, two, three, or four of these positions, arespecifically envisioned.

[0045] Variants also include polypeptides that differ in amino acidsequence due to mutagenesis. Variants that function as both GLP-1receptor agonists and glucagon receptor antagonists can be identified byscreening combinatorial libraries of mutants, for example, mutants ofpolypeptides with conservative substitutions at 1 or more positions(i.e., at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions) can be screenedfor GLP-1 receptor agonist activity and glucagon receptor antagonistactivity using methods well known in the art and described in thespecific examples below.

[0046] Analogs

[0047] An analog includes a propolypeptide, wherein the propolypeptideincludes an amino acid sequence of a polypeptide of the invention.Active polypeptides of the invention can be cleaved from the additionalamino acids in the propolypeptide molecule by natural, in vivo processesor by procedures well known in the art, such as by enzymatic or chemicalcleavage.

[0048] Fragments

[0049] A fragment is less than a full-length polypeptide of theinvention, including a full-length derivative, variant, or analog, whichhas GLP-1 receptor agonist and glucagon receptor antagonist activity.

[0050] Polynucleotides

[0051] Any polynucleotide sequence that encodes a polypeptide of theinvention can be used to express the polypeptide. Polynucleotides canconsist only of a coding sequence for a polypeptide or can includeadditional coding and/or non-coding sequences.

[0052] Polynucleotide sequences encoding a polypeptide of the inventioncan be synthesized in whole or in part using chemical methods well knownin the art (see, for example, Caruthers et al., Nuc. Acids Res. Symp.Ser. 215-23, 1980; Horn et al., Nucl. Acids Res. Symp. Ser. 225-32,1980). The polynucleotide that encodes the polypeptide can then becloned into an expression vector to express the polypeptide or into acloning vector, to propagate the polynucleotide.

[0053] As will be understood by those of skill in the art, it may beadvantageous to produce the polypeptide-encoding nucleotide sequencespossessing non-naturally occurring codons. For example, codons preferredby a particular prokaryotic or eukaryotic host can be selected toincrease the rate of polypeptide expression or to produce an RNAtranscript having desirable properties, such as a half-life, which islonger than that of a transcript generated from the naturally occurringsequence.

[0054] The nucleotide sequences disclosed herein can be engineered usingmethods generally known in the art to alter the polypeptide-encodingsequences for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe polypeptide or mRNA product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides canbe used to engineer the nucleotide sequences. For example, site-directedmutagenesis can be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

[0055] Vectors

[0056] The present invention also includes cloning and expressionvectors comprising one or more nucleotide sequences encoding apolypeptide of the invention. The nucleotide sequence can be inserted ina forward or reverse orientation. A DNA sequence may be inserted into avector by a variety of procedures. In general, a DNA sequence isinserted into an appropriate restriction endonuclease site by proceduresknown in the art and described in Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed., (Cold Spring Harbor, N.Y., 1989). Suchprocedures and others are deemed to be within the scope of those skilledin the art.

[0057] Examples of cloning vectors include, but are not limited topBR322, pUC18, pUC19, pSport, and pCRII.

[0058] In a preferred aspect of this embodiment, an expression vectorfurther comprises regulatory sequences, including, for example, apromoter, operably linked to the coding sequence. Large numbers ofsuitable expression vectors and promoters are known to those of skill inthe art and are commercially available. The following expression vectorsare provided by way of example. Bacterial expression vectors include,but are not limited to, pQE70, pQE60, pQE-9 (Qiagen), pBS, phagescript,psiXI74, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a(Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, PRIT5 (Pharmacia).Eukaryotic expression vectors include, but are not limited to, pWLneo,pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, PSVL(Pharmacia). However, any other cloning or expression vector may be usedas long as it is replicable and viable in the desired host. Promoterregions can be selected from any desired gene using CAT (chloramphenicoltransferase) expression vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include laci, lacZ, T3, T7, gpt, lambda PR, PL andtrp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of an appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0059] An expression vector also can contain a ribosome binding site fortranslation initiation, a transcription terminator, and appropriatesequences for amplifying expression. Expression vectors can contain agene to provide a phenotypic trait for selection of transformed hostcells, such as dihydrofolate reductase or neomycin resistance for aeukaryotic cell culture, or such as tetracycline or ampicillinresistance for culture in E. coli.

[0060] Libraries

[0061] In one embodiment, a library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A library of variants can be produced, forexample, by enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential variant amino acid sequences is expressible as individualpolypeptides or, alternatively, as a set of larger fusion proteins (forexample, for phage display) containing the set of sequences therein.

[0062] There are a variety of methods that can be used to producelibraries of potential variants from a degenerate oligonucleotidesequence. Chemical synthesis of a degenerate gene sequence can beperformed in an automatic DNA synthesizer, and the synthetic gene thenligated into an appropriate expression vector. Use of a degenerate setof genes allows for the provision, in one mixture, of all of thesequences encoding the desired set of potential analog sequences.Methods for synthesizing degenerate oligonucleotides are known in theart (see, e.g., Narang, Tetrahedron 39:3 (1983); Itakura et al., Annu.Rev. Biochem. 53:323 (1984); Itakura et al., Science 198:1056 (1984);Ike et al, Nucleic Acid Res. 11:477, 1983).

[0063] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis ofpolypeptides of the invention. The most widely used techniques, whichare amenable to high through-put analysis for screening large genelibraries, typically include cloning the gene library into replicableexpression vectors, transforming appropriate cells with the resultinglibrary of vectors, and expressing the combinatorial genes underconditions in which detection of a desired activity facilitatesisolation of the vector encoding the gene whose product was detected.Recursive ensemble mutagenesis (REM), a technique that enhances thefrequency of functional mutants in the libraries, can be used incombination with the screening assays to identify the desired variants.

[0064] Host Cells

[0065] The present invention also provides host cells containing theabove-described vectors. The host cell can be a higher eukaryotic cell,such as a mammalian cell, or a lower eukaryotic cell, such as a yeastcell. Alternatively, the host cell can be a prokaryotic cell, such as abacterial cell.

[0066] Host cells can be genetically engineered (transduced, transformedor transfected) with cloning or expression vectors of the invention. Thevector may be, for example, in the form of a plasmid, a viral particle,or a phage. Engineered host cells can be cultured in conventionalnutrient media modified as appropriate for activating promoters orselecting transformants. The selection of appropriate cultureconditions, such as temperature and pH, are well within the skill of theordinarily skilled artisan.

[0067] As representative examples of appropriate hosts, include, but arenot limited to, bacterial cells, such as E. coli, Salmonellatyphimurium, Streptomyces; fungal cells, such as yeast; insect cells,such as Drosophila S2 and Spodoptera Sf9; mammalian cells such as CHO,COS or Bowes melanoma. The selection of an appropriate host is deemed tobe within the scope of those skilled in the art from the teachingsherein.

[0068] Introduction of the construct into the host cell can be effected,for example, by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis et al., BASIC METHODS INMOLECULAR BIOLOGY, 1986). Constructs in host cells can be used in aconventional manner to produce the gene product encoded by therecombinant sequence.

[0069] Protein Expression

[0070] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described above and in Sambrook etal., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., (Cold SpringHarbor, N.Y., 1989).

[0071] Transcription of a DNA encoding polypeptides of the presentinvention by higher eukaryotes can be increased by inserting an enhancersequence into the expression vector. Enhancers are cis-acting elementsof DNA, usually from about 10 to 300 bp, that act on a promoter toincrease its transcription. Examples include the SV40 enhancer on thelate side of the replication origin (bp 100 to 270), a cytomegalovirusearly promoter enhancer, a polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers. Generally, recombinantexpression vectors will include origins of replication and selectablemarkers permitting transformation of the host cell, e.g., the ampicillinresistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoterderived from a highly expressed gene to direct transcription of adownstream structural sequence. Such promoters can be derived fromoperons encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), α factor, acid phosphatase, or heat shock proteins, among others.The heterologous structural sequence is assembled in appropriate phasewith translation, initiation and termination sequences, and preferably,a leader sequence capable of directing secretion of translated proteininto the periplasmic space or extracellular medium. Optionally, theheterologous sequence can encode a fusion protein including anN-terminal identification peptide imparting desired characteristics,e.g., stabilization or simplified purification of expressed recombinantproduct.

[0072] After transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isderepressed by appropriate means (e.g., temperature shift or chemicalinduction), and cells are cultured for an additional period. Cells aretypically harvested by centrifugation and disrupted by physical orchemical means. The resulting crude extract is retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents.

[0073] Various mammalian cell culture systems also can be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, C127, 3T3, CHO, HeLa and HBK celllines.

[0074] Protein Purification

[0075] Polypeptides of the present invention may be recovered andpurified from recombinant cell cultures by methods well known in theart, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxyapatite chromatography, and lectinchromatography. High performance liquid chromatography (HPLC) can beemployed as a final purification step.

[0076] Polypeptides of the invention can be conveniently isolated asdescribed in the specific examples below. A purified polypeptide is atleast about 70% pure, that is, the isolated polypeptide is substantiallyfree of cellular material and has less than about 30% (by dry weight) ofnon-polypeptide material. Preferably, the preparations are 85% through99% (i.e., 85, 87, 89, 91, 93, 95, 96, 97, 98, and 99%) pure. Purity ofthe preparations can be assessed by any means known in the art, such asSDS-polyacrylamide gel electrophoresis, mass spectroscopy and liquidchromatography.

[0077] Post-Translational Modification

[0078] Depending upon the host employed in a recombinant productionprocedure, polypeptides of the invention may be glycosylated withmammalian or other eukaryotic carbohydrates or may be non-glycosylated.Polypeptides of the invention may also include an initial methionineamino acid residue.

[0079] Chemical Synthesis

[0080] Alternatively, polypeptides of the invention can be producedusing chemical methods to synthesize its amino acid sequence, such as bydirect peptide synthesis using solid-phase techniques (see, for example,Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al,Science 269, 202-204, 1995). Polypeptide synthesis can be performedusing manual techniques or by automation. Automated synthesis can beachieved, for example, using Applied Biosystems 431A Peptide Synthesizer(Perkin Elmer). Optionally, fragments of a polypeptide can be separatelysynthesized and combined using chemical methods to produce a full-lengthmolecule.

[0081] A newly synthesized polypeptide can be substantially purified bypreparative high performance liquid chromatography (see, for example,Creighton, Proteins: Structures And Molecular Principles, W H Freemanand Co., New York, N.Y., 1983). The composition of a syntheticpolypeptide of the present invention can be confirmed by amino acidanalysis or sequencing by, for example, the Edman degradation procedure(see, Creighton, supra). Additionally, any portion of the amino acidsequence of the polypeptide can be altered during direct synthesisand/or combined using chemical methods with sequences from otherproteins to produce a variant polypeptide or a fusion polypeptide.

[0082] Pharmaceutical Applications

[0083] Polypeptides of the present invention can be used to treat type 2diabetes (non-insulin dependent diabetes mellitus) and/or to preventsubjects with impaired glucose tolerance, impaired fasting glucose,hypoglycemia, and/or obesity from developing type 2 diabetes.

[0084] Pharmaceutical Compositions

[0085] Polypeptides of the present invention may be combined with asuitable pharmaceutical carrier to form a pharmaceutical composition forparenteral administration. Such compositions comprise a therapeuticallyeffective amount of the polypeptide and a pharmaceutically acceptablecarrier or excipient. Pharmaceutically acceptable carriers, include butare not limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The formulation should suit the modeof administration.

[0086] Pharmaceutical compositions may be administered in a convenientmanner, e.g., by oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal, or intradermal routes.Pharmaceutical compositions are administered in an amount, that iseffective for treating and/or prophylaxis of the specific indication.Suitable doses range from at least about 3.5 ng of active polypeptide(i.e., not including the weight of a PEG or fatty acid moiety)/kg bodyweight to about 100 μg/kg body weight per day. In most cases, the dosageis from about 0.1 μg/kg to about 35 μg/kg (i.e., 0.1, 1, 5, 10, 15, 20,25, 30, and 35 μg/kg) body weight daily, taking into account the routesof administration, symptoms, etc. These numbers do not take into accountthe bioavailability of the peptide in vivo, in which case more or lessmay be used to attain the effective dose desired. Determination of adose is well within the skill of the ordinary artisan and requires onlyroutine screening.

[0087] Methods of Use

[0088] Polypeptides of the present invention may also be employed incombination with other pharmaceutical agents useful in the treatment ofdiabetes, impaired fasting glucose, impaired glucose tolerance,hyperglycemia, and obesity. Suitable agents include insulinsecretagogues, insulin sensitizers, and metformin HCl.

[0089] The present invention includes methods for the treatment ofdiabetes and related diseases and conditions. One such method comprisesthe step of administering to a subject in need thereof, atherapeutically effective amount of one or more polypeptides of theinvention.

[0090] Polypeptides of the invention may be used in methods of theinvention to treat diseases, such as diabetes, including Type 2diabetes. Such methods may also delay the onset of diabetes and diabeticcomplications. Other diseases and conditions that may be treated orprevented using polypeptides of the invention in methods of theinvention include: Maturity-Onset Diabetes of the Young (MODY) (Herman,et al., Diabetes 43:40 (1994)), Latent Autoimmune Diabetes Adult (LADA)(Zimmet, et al., Diabetes Med. 11:299 (1994)), impaired glucosetolerance (IGT) (Expert Committee on Classification of DiabetesMellitus, Diabetes Care 22 (Supp. 1) S5 (1999)), impaired fastingglucose (IFG) (Charles, et al., Diabetes 40:796 (1991)), gestationaldiabetes (Metzger, Diabetes, 40:197 (1991), and metabolic syndrome X.

[0091] Polypeptides of the invention may also be used in methods of theinvention to treat secondary causes of diabetes (Expert Committee onClassification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1), S5(1999)). Such secondary causes include glucocorticoid excess, growthhormone excess, pheochromocytoma, and drug-induced diabetes. Drugs thatmay induce diabetes include, but are not limited to, pyriminil,nicotinic acid, glucocorticoids, phenytoin, thyroid hormone,β-adrenergic agents, α-interferon and drugs used to treat HIV infection.

[0092] Polypeptides of this invention may also be useful for thetreatment of bulimia and obesity including associated dyslipidemia andother obesity- and overweight-related complications such as, forexample, cholesterol gallstones, cancer (e.g., colon, rectum, prostate,breast, ovary, endometrium, cervix, gallbladder, and bile duct),menstrual abnormalities, infertility, polycystic ovaries,osteoarthritis, and sleep apnea, as well as for a number of otherpharmaceutical uses associated therewith, such as the regulation ofappetite and food intake, dyslipidemia, hypertriglyceridemia,atherosclerotic diseases such as heart failure, hyperlipidemia,hypercholesteremia, low HDL levels, hypertension, cardiovascular disease(including atherosclerosis, coronary heart disease, coronary arterydisease, and hypertension), cerebrovascular disease and peripheralvessel disease. The polypeptides of this invention may also be usefulfor treating physiological disorders related to, for example, regulationof insulin sensitivity, inflammatory response, plasma triglycerides,HDL, LDL, and cholesterol levels and the like.

[0093] The methods and polypeptides of the present invention may be usedalone or in combination with additional therapies and/or compounds knownto those skilled in the art in the treatment of diabetes and relateddisorders. Alternatively, the methods and polypeptides described hereinmay be used, partially or completely, in combination therapy.

[0094] Polypeptides of the invention may also be administered incombination with other known therapies for the treatment of diabetes,including PPAR agonists, sulfonylurea drugs, non-sulfonylureasecretagogues, α-glucosidase inhibitors, insulin sensitizers, insulinsecretagogues, hepatic glucose output lowering compounds, insulin andanti-obesity drugs. Such therapies may be administered prior to,concurrently with or following administration of the compound of theinvention. Insulin includes both long and short acting forms andformulations of insulin. PPAR agonist may include agonists of any of thePPAR subunits or combinations thereof. For example, PPAR agonist mayinclude agonists of PPAR-α, PPAR-γ, PPAR-67 or any combination of two orthree of the subunits of PPAR. PPAR agonists include, for example,rosiglitazone and pioglitazone. Sulfonylurea drugs include, for example,glyburide, glimepiride, chlorpropamide, and glipizide. α-glucosidaseinhibitors that may be useful in treating diabetes when administeredwith a compound of the invention include acarbose, miglitol andvoglibose. Insulin sensitizers that may be useful in treating diabeteswhen administered with a compound of formula (I) includethiozolidinediones and non-thiozolidinediones. Hepatic glucose outputlowering compounds that may be useful in treating diabetes whenadministered with a compound of the invention include metformin, such asGlucophage and Glucophage XR. Insulin secretagogues that may be usefulin treating diabetes when administered with a compound of the inventioninclude sulfonylurea and non-sulfonylurea drugs: GLP-1, GIP, PAC/VPACreceptor agonists, secretin, nateglinide, meglitinide, repaglinide,glibenclamide, glimepiride, chlorpropamide, glipizide. GLP-1 includesderivatives of GLP-1 with longer half-lives than native GLP-1, such as,for example, fatty-acid derivatized GLP-1 and exendin. In one embodimentof the invention, polypeptides of the invention are used in combinationwith insulin secretagogues to increase the sensitivity of pancreaticbeta cells to the insulin secretagogue.

[0095] Polypeptides of the invention may also be used in methods of theinvention in combination with anti-obesity drugs. Anti-obesity drugsinclude P-3 agonists, CB-1 antagonists, appetite suppressants, such as,for example, sibutramine (Meridia), and lipase inhibitors, such as, forexample, orlistat (Xenical).

[0096] Polypeptides of the invention may also be used in methods of theinvention in combination with drugs commonly used to treat lipiddisorders in diabetic patients. Such drugs include, but are not limitedto, HMG-CoA reductase inhibitors, nicotinic acid, bile acidsequestrants, and fibric acid derivatives. Polypeptides of the inventionmay also be used in combination with anti-hypertensive drugs, such as,for example, β-blockers and ACE inhibitors.

[0097] Such co-therapies may be administered in any combination of twoor more drugs (e.g., a compound of the invention in combination with aninsulin sensitizer and an anti-obesity drug). Such co-therapies may beadministered in the form of pharmaceutical compositions, as describedabove.

[0098] Kits

[0099] The invention also provides pharmaceutical packs or kitscomprising one or more containers filled with one or more of theingredients of pharmaceutical compositions of the invention. A notice ina form prescribed by a governmental agency that regulates themanufacture, use or sale of pharmaceuticals or biological products andreflecting approval by the agency can be associated with the pack orkit.

[0100] Gene Therapy

[0101] A polypeptide of the invention may also be expressed in vivo,which is often referred to as “gene therapy.” Thus, for example, cellsmay be engineered with a polynucleotide (DNA or RNA) encoding for thepolypeptide ex vivo and the engineered cells then provided to a patientto be treated with the polypeptide. Such methods are well known in theart. For example, cells may be engineered by procedures known in the artby use of a retroviral particle containing RNA encoding for polypeptidesof the present invention.

[0102] In a preferred embodiment, the DNA encoding the polypeptides ofthe invention is used in gene therapy for disorders such as diabetes.According to this embodiment, gene therapy with DNA encodingpolypeptides of the invention is provided to a patient in need thereof,concurrent with, or immediately after diagnosis.

[0103] Local delivery of polypeptides using gene therapy may provide thetherapeutic agent to the target area, i.e., the pancreas. For instance apancreas-specific promoter was used to create a beta-cell pancreatictumor mouse model (Hanahan, Nature 315(6015):115-22, 1985).

[0104] Both in vitro and in vivo gene therapy methodologies arecontemplated. Several methods for transferring potentially therapeuticgenes to defined cell populations are known. See, e.g., Mulligan,Science 260: 926-31, 1993. These methods include:

[0105] 1) Direct gene transfer. See, e.g., Wolff et al., “Direct Genetransfer Into Mouse Muscle In Vivo,” Science 247:1465-68, 1990;

[0106] 2) Liposome-mediated DNA transfer. See, e.g., Caplen et al.,“Liposome-mediated CFTR Gene Transfer To The Nasal Epithelium OfPatients With Cystic Fibrosis,” Nature Med. 3: 39-46, 1995; Crystal,“The Gene As A Drug,” Nature Med. 1:15-17, 1995; Gao and Huang, “A NovelCationic Liposome Reagent For Efficient Transfection Of MammalianCells,” Biochem. Biophys. Res. Comm. 179:280-85, 1991;

[0107] 3) Retrovirus-mediated DNA transfer. See, e.g., Kay et al., “InVivo Gene Therapy Of Hemophilia B: Sustained Partial Correction InFactor IX-Deficient Dogs,” Science 262:117-19, 1993; Anderson, “HumanGene Therapy,” Science 256:808-13, 1992.

[0108] 4) DNA Virus-mediated DNA transfer. Such DNA viruses includeadenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes viruses(preferably herpes simplex virus based vectors), and parvoviruses(preferably “defective” or non-autonomous parvovirus based vectors, morepreferably adeno-associated virus based vectors, most preferably AAV-2based vectors). See, e.g., Ali et al., “The Use Of DNA Viruses AsVectors For Gene Therapy,” Gene Therapy 1:367-84,1994; U.S. Pat. No.4,797,368, and U.S. Pat. No. 5,139,941.

[0109] Gene Therapy Vector Systems

[0110] The choice of a particular vector system for transferring a geneof interest will depend on a variety of factors. One important factor isthe nature of the target cell population. Although retroviral vectorshave been extensively studied and used in a number of gene therapyapplications, these vectors are generally unsuited for infectingnon-dividing cells. In addition, retroviruses have the potential foroncogenicity. However, recent developments in the field of lentiviralvectors may circumvent some of these limitations. See Naldini et al.,Science 272:263-7, 1996.

[0111] The skilled artisan will appreciate that any suitable genetherapy vector encoding polypeptides of the invention can be used inaccordance with this embodiment. The techniques for constructing suchvectors are known. See, e.g., Anderson, Nature 392 25-30, 1998; Vermaand Somia, Nature 389 239-242, 1998. Introduction of the vector to thetarget site may be accomplished using known techniques.

[0112] Suitable gene therapy vectors include one or more promoters.Suitable promoters which may be used include, but are not limited to,viral promoters (e.g., retroviral LTR, SV40 promoter, adenovirus majorlate promoter, respiratory syncytial virus promoter, B19 parvoviruspromoter, and human cytomegalovirus (CMV) promoter described in Milleret al, Biotechniques 7(9): 980-990, 1989), cellular promoters (e.g.,histone, pol III, and β-actin promoters), and inducible promoters (e.g.,MMT promoter, metallothionein promoter, and heat shock promoter). Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0113] Retroviruses

[0114] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0115] The retroviral plasmid vector is used to transduce packaging celllines to form producer cell lines. Examples of packaging cells whichmaybe transfected include, but are not limited to, the PE501, PA317,ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E−86,GP+envAm12,and DAN cell lines as described in Miller, Human Gene Therapy, 1: 5-14,1990. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host. Theproducer cell line generates infectious retroviral vector particles thatinclude the nucleic acid sequence(s) encoding polypeptides of theinvention. Such retroviral vector particles then may be used, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encodingpolypeptides of the invention. Eukaryotic cells that can be transducedinclude, but are not limited to, embryonic stem cells, embryoniccarcinoma cells, as well as hematopoietic stem cells, hepatocytes,fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchialepithelial cells.

[0116] Adenoviruses and Adeno-Associated Viruses

[0117] Adenoviruses have the advantage that they have a broad hostrange, can infect quiescent or terminally differentiated cells, such asneurons or hepatocytes, and appear essentially non-oncogenic. See, e.g.,Ali et al., 1994, at page 367. Adenoviruses do not appear to integrateinto the host genome. Because they exist extrachromosomally, the risk ofinsertional mutagenesis is greatly reduced. Ali et al., 1994, at page373.

[0118] Adeno-associated viruses exhibit similar advantages asadenoviral-based vectors. However, AAVs exhibit site-specificintegration on human chromosome 19 (All et al., 1994, at page 377).

[0119] Transkaryotic Therapy

[0120] A different approach to gene therapy is “transkaryotic therapy”wherein the patient's cells are treated ex vivo to induce the dormantchromosomal genes to produce the protein of interest afterreintroduction to the patient. Transkaryotic therapy assumes theindividual has a normal complement of genes necessary for activation.Transkaryotic therapy involves introducing a promoter or other exogenousregulatory sequence capable of activating the nascent genes, into thechromosomal DNA of the patients' cells ex vivo, culturing and selectingfor active protein-producing cells, and then reintroducing the activatedcells into the patient with the intent that they then become fullyestablished. The “gene activated” cells then manufacture the protein ofinterest for some significant amount of time, perhaps for as long as thelife of the patient. U.S. Pat. Nos. 5,641,670 and 5,733,761 disclose indetail this concept.

[0121] In order that this invention may be better understood, thefollowing examples are set forth. These examples are for the purpose ofillustration only, and are not to be construed as limiting the scope ofthe invention in any manner. All patents, patent applications, andreferences cited in this disclosure are incorporated herein by referencein their entirety.

EXAMPLES Example 1

[0122] Peptide Synthesis Methodology

[0123] The following general procedure was followed to synthesize someof polypeptides of the invention. Peptide synthesis was carried out bythe FMOC/t-Butyl strategy (Peptide Synthesis Protocols (1994), Volume 35by Michael W. Pennington & Ben M. Dunn) under continuous flow conditionsusing Rapp-Polymere PEG-Polystyrene resins (Rapp-Polymere, Tubingen,Germany). At the completion of synthesis, peptides were cleaved from theresin and de-protected using TFA/DTT/H₂O/Triisopropyl silane (88/5/5/2).Peptides were precipitated from the cleavage cocktail using cold diethylether. The precipitate was washed three times with the cold ether andthen dissolved in 5% acetic acid prior to lyophilization. Peptideidentity was confirmed by reversed-phase chromatography on a YMC-PackODS-AQ column (YMC, Inc., Wilmington, N.C.) on a Waters ALLIANCE® system(Waters Corporation, Milford, Ma.) using water/acetonitrile with 3% TFAas a gradient from 0% to 100% acetonitrile, and by MALDI massspectrometry on a VOYAGER DE™ MALDI Mass Spectrometer, (model 5-2386-00,PerSeptive BioSystems, Framingham, Ma.). Matrix buffer (50/50dH₂O/acetonitrile with 3% TFA) peptide sample was added to Matrix buffer1/1. Those peptides not meeting the purity criteria of >95% werepurified by reversed-phase chromatography on a Waters Delta Prep 4000HPLC system (Waters Corporation, Milford, Ma.).

Example 2

[0124] Peptide Cloning

[0125] To establish a robust method for expressing polypeptides of theinvention and mutants thereof, nucleotide sequences encodingpolypeptides were cloned C-terminal to GST with a single Factor Xarecognition site separating the monomeric peptide and GST. The geneencoding the Factor Xa recognition site fused to the DNA sequence of thepeptide to be produced has been synthesized by hybridizing twooverlapping single-stranded DNA fragments (70-90mers) containing a BamHIor EcoRI restriction enzyme site immediately 5′ to the DNA sequence ofthe polynucleotide to be cloned, followed by DNA synthesis of theopposite strands via the large fragment of DNA polymerase I (LifeTechnologies, Inc., Gaithersburg, Md.).

[0126] The DNA sequence chosen for each polynucleotide was based on thereverse translation of the designed amino acid sequence of each peptide.In some cases, the polynucleotide encoding the peptide is generated byPCR mutagenesis (Picard et al, Nucleic Acids Res 22: 2587-91, 1994;Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed.,Cold Spring Harbor Laboratory Press, New York) of a polynucleotidealready made by the method described above. The double-stranded productis then digested by BamHI and EcoRI and ligated into pGEX-6P-1 (AmershamPharmacia Biotech) which has also been cleaved by BamHI and EcoRI.

[0127] For example, when the DNA sequence the polypeptide identified asSEQ ID NO: 7 was cloned into pGEX-6P-1, the following polypeptidesequence was expressed as fusions with glutathione S-transferase (GST):IEGRHSQGTFTSDYAKYLDARRAKEFIAWLVKGRG (SEQ ID NO: 33), where the first 4amino acids, IEGR, is the factor Xa recognition site and the remaining31 amino acids is the amino acid sequence identified as SEQ ID NO: 7.

[0128] The first and last six nucleotides of the nucleic acid sequenceencoding the polypeptide identified as SEQ ID NO: 7 represent therestriction enzyme sites (BamHI and EcoRI) used for cloning into thepGEX6P-1 plasmid (FIG. 1). The “TAATGA” sequence immediately precedingthe EcoRI site (“GAATTC”) encodes two stop codons. The rest of the DNAsequence encodes the Factor Xa-polypeptide identified as SEQ ID NO: 7fusion sequence.

Example 3

[0129] Peptide Recombinant Expression and Purification

[0130] BL21 (DE3) cells (Stratagene) transformed with a GST-peptidefusion containing plasmids were grown at 37° C. until OD₆₀₀ reached 0.6to 1.0 and induced by 1 mM IPTG (Life Technologies) for 2 hours at 37°C. Two liters of cells were spun at 7,700×g for 15 minutes, and storedat −20° C. The cell pellet was resuspended in 80 ml B-PER bacterialprotein extraction reagent (Cat No. 78248, Pierce) and 1× CompleteProtease Inhibitor (Roche) until the cell suspension was homogenous. Thehomogenous mixture was gently shaken at room temperature for 10 minutes.Lysosome and Dnase I were added to a final concentration of 200 μg/mland 10 μg/ml to further solubilize proteins and to reduce viscosity,respectively. The mixture was incubated for an additional 5 minutes.Cellular debris was spun down at 27,000 g for 20 minutes. Thesupernatant was mixed with 2 mL of pre-washed Glutathione Sepharose 4Bresin (Pharmacia) on a shaker overnight at 4° C. The resin was spun downat 1,500 g for 15 minutes, packed into empty Poly-Prep ChromatographyColumns (Bio-Rad), washed with 30 mL PBS followed by 10 mL of Factor Xabuffer (1 mM CaCl₂, 100 mM NaCl, and 50 mM Tris-HCl, pH 8.0). Thepeptides were cleaved off the column with 60 units of Factor Xa(Pharmacia) in 1 mL of Factor Xa buffer, overnight at 4 ° C. Theseeluants were then run on a C₁₈ HPLC (Beckman System Gold), using a 2 mLloop and flow rate of 1 mL/min with the following program: 5 minutes ofBuffer A (0.1% TFA/H₂O), 30 minutes of gradient to 100% Buffer B (0.1%TFA/ACN), 10 minutes of Buffer A, 10 minutes of gradient, and 10 minutesof Buffer A. Peak fractions (0.5 mL each) were collected and screened by10-20% Tricine-SDS gel electrophoresis. The identity of each peptide wasconfirmed by mass spectrometry using the mass predicted from the aminoacid sequence. Typical yields are approximately 50 μg free peptides perliter of E. coli culture. Recombinant peptides have been shown to havethe same activities as their synthetic versions.

[0131] The following table, Table 2, contains some selected polypeptidesmade according to the peptide synthesis protocol discussed above(Example 1), or recombinantly as described above. TABLE 2 GlucagonHSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO:1) GLP-1 (7-36)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 (SEQ ID NO:2) GLP-1 (7-37)HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO:3) G1HSQGTFTSDYSKYLDSRRAQDFVQWLVKGR-NH2 (SEQ ID NO:4) G5HSQGTFTSDYSKYLEGQAAKEFIAWLVKGR-NH2 (SEQ ID NO:5) G27HSQGTFTSDYAKYLDARRAKEFIAWLVKGR-NH2 (SEQ ID NO:6) G51HSQGTFTSDYAKYLDARRAKEFIAWLVKGRG (SEQ ID NO:7) G55HSQGTFTSDYARYLDARRAKEFIAWLVKGR-NH2 (SEQ ID NO:8) G56HSQGTFTSDYAAYLDARRAKEFIAWLVKGR-NH2 (SEQ ID NO:9) G57HSQGTFTSDYAKYLDAARAKEFIAWLVKGR-NH2 (SEQ ID NO:10) G58HSQGTFTSDYAKYLDAKKAKEFIAWLVKGRG (SEQ ID NO:11) G59HSQGTFTSDYARYLDAKKAKEFIAWLVKGRG (SEQ ID NO:12) G60HSQGTFTSDYAKYLDAAKAKEFIAWLVKGRG (SEQ ID NO:13) G61HSQGTFTSDYARYLDAAKAKEFIAWLVKGRG (SEQ ID NO:14) G71HSQGTFTSDYAKYLDARRACEFIAWLVKGRG (SEQ ID NO:15) G74HSQGTFTSDYAKYLDARRAKEFIAWLVCGRG (SEQ ID NO:16) G75HSQGTFTSDYAKYLDARRAKEFIAWLVKCRG (SEQ ID NO:17) G76HSQGTFTSDYAKYLDARRAKEFIAWLVKGCG (SEQ ID NO:18) G77HSQGTFTSDYAKYLDARRAKEFIAWLVKGRC (SEQ ID NO:19) G277HSQGTFTSDYAKYLDARRAKEFIAWLVKGRC(22 kD) (SEQ ID NO:20) G82HSQGTFTSDYARYLDARRAKEFIAWLVRGRG (SEQ ID NO:21) G83HSQGTFTSDYARYLDARRAREFIKWLVRGRG (SEQ ID NO:22) G84HSQGTFTSDYARYLDARRAREFIAWLVKGRG (SEQ ID NO:23) G85HSQGTFTSDYARYLDARRAREFIAWLVRGRGK (SEQ ID NO:24) G185HSQGTFTSDYARYLDARRAREFIKWLVRGRC (SEQ ID NO:25) G2185HSQGTFTSDYARYLDARRAREFIKWLVRGRC(22 kD) (SEQ ID NO:26) G4185HSQGTFTSDYARYLDARRAREFIKWLVRGRC(43 kD) (SEQ ID NO:27) G87HSQGTFTSDYAKYLDARRAK(FA)EFIAWLVKGR-NH2 (SEQ ID NO:28) G88HSQGTFTSDYAKYLDARRAKEFIAWLVKGRK(FA) (SEQ ID NO:29) G90HSQGTFTSDYARYLDARRAREFIK(FA)WLVRGRG (SEQ ID NO:30) G182HSQGTFTSDYARYLDARRAREFIKWLVRGRGK(FA) (SEQ ID NO:31) G183HSQGTFTSDYARYLDARRAK(FA)EFIKWLVRGRG (SEQ ID NO:32)

Example 4

[0132] Preparation of RINm5F Cell Membranes

[0133] Flasks of RINm5F cells were washed with PBS, scraped in 20 mMHepes-1 mM EDTA-250 mM sucrose buffer containing protease inhibitors(HES) and homogenized in a dounce homogenizer followed by repeatedresuspension through a 23 gauge needle. Unbroken cells and nuclei wereremoved by centrifugation at 500×g for 5 minutes. The pellet wasresuspended in HES buffer using a 23 gauge needle and the centrifugationwas repeated. The supernatants from the two spins were combined andcentrifuged at 40,000×g for 20 min. The resulting plasma membrane pelletwas resuspended in HES buffer using a 23 gauge needle followed by a 25gauge needle. Membranes were stored at −80° C. until use.

Example 5

[0134] Preparation of Plasma Membranes from Rat Liver

[0135] Rats were sacrificed and livers removed into ice cold TES buffer(20 mM Tris, pH 7.5, 1 mM EDTA, 255 mM sucrose containing proteaseinhibitors). The wet weight of the livers was determined and the tissueminced in 5 volumes of ice cold TES buffer and a slurry prepared using apolytron. All buffers and spins were kept at 4° C. The slurry wasfurther homogenized using three strokes of a handheld douncehomogenizer. The homogenate was passed through several layers ofcheesecloth and spun at 25,000×g for 10 minutes. Pellets werehomogenized in 22.1 ml TES buffer using 10 strokes of a handheld douncehomogenizer. Twenty-seven point nine milliliters of 2.4 M sucrose in TEwas added and mixed well. The homogenate was distributed among severaltubes, and each tube was overlaid with 7 ml of TES buffer. The tubeswere spun at 120,000×g for 60 minutes. The plasma membranes were removedfrom the interface that formed during the spin, diluted in TES andconcentrated using a 15 minute spin at 200,000×g. The resulting pelletwas again homogenized in 9.8 ml TES buffer, combined with 10.3 ml 2.4Msucrose, overlaid with 7 ml TES, and centrifuged at 180,000×g for 30min. The plasma membranes were removed from the interface, diluted withTES buffer, and collected by centrifugation at 200,000×g for 15 min. Thefinal pellet was resuspended in TES. Plasma membranes were stored at−80° C. until ready for use.

Example 6

[0136] Preparation of Rat Hepatocytes

[0137] Hepatocytes were isolated according to a modified procedure ofBerry and Friend (J. Cell. Biol. 43:506, 1969). A fed male Wistar ratwas anesthetized with sodium pentobarbital (55 mg/kg, i.p.). The rat wasplaced on its back on the tray of the liver perfusion apparatus (37°C.), and its limbs were secured with laboratory tape. The ventriclesurface was cleaned with 70% alcohol, and the abdominal cavity wasopened with scissors to expose the liver, portal vein, hepatic arteryand the posterior vena cava. Ligatures were placed loosely around thethree vessels. A cannula was inserted into the portal vein and wassecured with the ligature. The hepatic artery and the posterior venacava below the liver were tied off with the ligatures. The perfusionpump was turned on and the descending aorta was immediately severed toallow the buffer to escape and to exsanguinate the rat. The chest cavitywas quickly opened to expose the heart. A cannula was inserted into thesuperior vena cava through the heart and was secured with a ligature tothe complete the circulation of buffer through the liver. The liver wasperfused with Krebs-Henseleit buffer containing 5-10% sheep RBC andunder constant 95% O₂/5% CO₂ flow in situ at a flow rate of 14ml/minute. It was perfused in a non-circulating system for 5 min andthen followed by a re-circulating system (with 30 mg collagenase) foranother 30 minutes. The liver was then removed, minced with scissors ina plastic beaker and filtered through a nylon sieve. The hepatocyteswere separated from debris by a series of buffer washings andcentrifugations (690 rpm, room temperature, 90 sec). An aliquot of cellswas diluted with buffer, stained with 0.4% Trypan Blue and counted. Therest of the cells were diluted with buffer to a density appropriate foreach assay.

Example 7

[0138] Protocol for Rat Islet Isolation

[0139] Sprague Dawley rats (275-320 g) were used as the source of donorislets. Briefly, the pancreas was filled with 10 ml of coldreconstituted Liberase RI (Boehringer Manheim), harvested and incubatedwith additional 5 ml enzyme solution in water bath for 30 minutes.Tissue suspension was washed twice with cold 10% FBS/Hanks buffer(Gibco), resuspended in 8 ml 25% ficoll (Sigma) and then layered with 5ml each of 23%, 20% and 11% ficoll. The islets in the 20% layer aftercentrifugation were removed, washed twice with cold 10% FBS/Hank bufferand resuspended in 10% FBS/RPMI 1640 media (Sigma).

Example 8

[0140] Competitive Binding of Peptide to the GLP-1 Receptor in RINm5FCell Plasma Membranes

[0141] IC₅₀ values for the competitive binding of polypeptides andpolypeptide fragments, variants, and analogs of the invention to theGLP-1 receptor in RINm5F membranes typically are at least about 0.01 nMup to about 20 nM (i.e., 0.01, 0.11, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 nM). IC₅₀ values for polypeptidederivatives of the invention typically are at least 0.01 nM up to about500 nM (i.e., 0.01, 0.1, 1, 10, 50, 100, 150, 200, 250, 300, 350, 400,450, or 500 nM).

[0142] Competitive binding of some polypeptides of the invention toRINm5F cell plasma membranes was measured as follows. Ninety-six-wellGF/C filtration plates (Millipore, Bedford, Ma.) were blocked with 0.3%PEI for at least one hour and washed twice with binding bufferconsisting of 20 mM Tris, 2 mM EDTA, pH 7.5, 1 mg/ml BSA, and 1 mg/mlbacitracin. Five micrograms of RINm5F cell plasma membranes diluted inbinding buffer were applied to each well together with 0.05 μCi ¹²⁵Ilabeled GLP-1 and peptide concentrations ranging from 1×10⁻¹² to 1×10⁻⁵M. Following a 60 minute incubation at room temperature, the plates werewashed 3 times with ice-cold PBS containing 1 mg/ml BSA. The plates weredried, scintillant was added to each well, and cpm per well determinedusing a Wallac Microbeta counter.

[0143] The number of ¹²⁵I counts bound to the membranes at eachconcentration of peptide were plotted and analyzed by nonlinearregression using Prizm software to determine the IC₅₀. The polypeptidesdisclosed in Table 2 bound to the GLP-1 receptor present in the plasmamembranes isolated from RINm5F cells with IC₅₀ values of between 1.4 nMand 248 nM (determined from a minimum of three trials). IC₅₀ is theconcentration of a polypeptide at which maximal binding of labeled GLP-1(7-36) (SEQ ID NO: 2) is reduced by 50%.

Example 9

[0144] Competitive Binding of Peptide to the Glucagon Receptor in RatLiver Plasma Membranes

[0145] IC₅₀ values for the competitive binding of polypeptides andpolypeptide fragments, derivatives, variants, and analogs of theinvention to the glucagon receptor in rat liver membranes typically areat least about 0.1 nM up to about 1000 nM (i.e., 0.1 1, 10, 50, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, or 1000 nM).

[0146] Competitive binding of some polypeptides of the invention to ratliver plasma membranes was measured as follows. Ninety-six-well GF/Cfiltration plates (Millipore, Bedford, Ma.) were blocked with 0.1% PEIfor at least one hour and washed twice with binding buffer consisting of20 mM Tris, 2 mM EDTA, pH 7.5, 1 mg/ml BSA, and 1 mg/ml bacitracin.Three micrograms of rat liver plasma membranes diluted in binding bufferwere applied per well together with 0.05 μCi ¹²⁵I labeled glucagon andpeptide concentrations ranging from 1×10⁻¹² to 1×10⁻⁵ M. Following a 60minute incubation at room temperature, the plates were washed 3 timeswith ice-cold PBS containing 1 mg/ml BSA. The plates were dried,scintillant was added to each well, and cpm per well determined using aWallac Microbeta counter.

[0147] The number of ¹²⁵I counts bound to the membranes at eachconcentration of peptide were plotted and analyzed by nonlinearregression using Prizm software to determine the IC₅₀. The polypeptidesdisclosed in Table 2 bound to the glucagon receptor present in theplasma membranes isolated from rat liver with IC₅₀ values of between11.7 nM and 726 nM (determined from a minimum of three trials). IC₅₀ isthe concentration of a polypeptide at which maximal binding of labeledglucagon is reduced by 50%.

Example 10

[0148] Measurement of Peptide Signaling Through GLP-1 Receptor UsingCyclic AMP Scintillation Proximity Assay (SPA)

[0149] For polypeptides and polypeptide fragments, variants, and analogsof the invention, “activation” of the GLP-1 receptor in a cAMPscintillation proximity assay, is induction of a maximal activity thatis at least about 80% up to about 200% (i.e., 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, or 200%) of the maximal activity induced by the nativeGLP-1(7-36) (SEQ ID NO: 2) with a relative potency of at least 4% up toabout 1000% (i.e., 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900, or 1000%). For polypeptidederivatives of the invention, “activation” of the GLP-1 receptor in acAMP scintillation proximity assay is induction of a maximal activitythat is at least about 80% up to about 200% (i.e., 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, or 200%) of the maximal activity induced by thenative GLP-1(7-36) (SEQ ID NO: 2) with a relative potency of at least0.5% up to about 1000% (i.e., 0.5, 1, 10, 50, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000%). “Relative potency” is the EC₅₀ of nativeGLP-1(7-36) (SEQ ID NO: 2) divided by the EC₅₀ of a polypeptide of theinvention, multiplied by 100. “EC₅₀” is the concentration of apolypeptide at which 50% of the maximal activity is achieved.

[0150] Peptide signaling of GLP-1 receptor for some polypeptides of theinvention using cAMP scintillation proximity assay was measured asfollows. RINm5F cells were plated in 96-well plates (Costar) at1.5×10⁵cells/well and grown at 37° C. for 24 hours in RPMI 1640, 5% FBS,antibiotic/antimycotic (Gibco BRL). The media was removed and the cellswere washed twice with PBS. The cells were incubated with peptideconcentrations ranging from 1×10 ⁻² to 1×10 ⁻⁵ M in Hepes-PBS containing1% BSA and 100 μM IBMX for 15 min at 37° C. For assay of peptidesconjugated with fatty acid, the BSA was omitted from the incubationbuffer. The incubation buffer was removed, and the cells were lysed inthe lysis reagent provided with the cAMP Scintillation Proximity Assay(SPA) direct screening assay system (Amersham Pharmacia Biotech Inc,Piscataway, N.J.). The amount of cAMP (in pmol) present in the lysateswas determined following instructions provided with this kit.

[0151] The amount of cAMP (in pmol) produced at each concentration ofpeptide was plotted and analyzed by nonlinear regression using Prizmsoftware to determine the EC₅₀ for each peptide. The average relativepotency value for GLP-1 receptor activation for the polypeptidesdisclosed in Table 2 was between 0.6% and 76.1%. The maximum activityinduced ranged from 83% to 132% of the native peptide GLP-1(7-36) (SEQID NO: 2) (determined from a minimum of three trials). See Table 4.

Example 11

[0152] Measurement of Peptide Signaling Through Glucagon Receptor UsingCyclic AMP Scintillation Proximity Assay (SPA)

[0153] For polypeptides and polypeptide fragments, variants, and analogsof the invention, “activation” of the glucagon receptor in a cAMPscintillation proximity assay is induction of a maximal activity that isat least about 0% up to about 75% (i.e., 0, 10 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, or 75%) of the maximal activity induced by thenative glucagon (SEQ ID NO: 1) with a relative potency from at leastabout 0.001 to about 5% (i.e., 0.001, 0.01, 0.1, 1, 2, 3, 4, or 5%). Forpolypeptide derivatives of the invention, “activation” of the glucagonreceptor in a cAMP scintillation proximity assay is induction of amaximal activity that is at least about 0% to about 40% (i.e., 0, 1, 10,20, 30, or 40%) of the maximal activity induced by the native glucagon(SEQ ID NO: 1) with a relative potency of at least about 0.001 to about1% (i.e., 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or1%). “Relative potency” is the EC₅₀ of native glucagon (SEQ ID NO: 1)divided by the EC₅₀ of a polypeptide of the invention, multiplied by100. “EC₅₀” is the concentration of a polypeptide at which 50% of themaximal activity is achieved.

[0154] Peptide signaling of glucagon receptor for some polypeptides ofthe invention using cAMP scintillation proximity assay was measured asfollows. Freshly isolated rat hepatocytes were plated in 96 well platesat 7.5×10⁴ or 2×10⁵ cells/well in Hepes-bicarbonate-PBS containing 1%BSA and 100 μM IBMX. Following equilibration at 37° C. in a 5% CO₂/95%O₂environment for 10 min, peptide at concentrations ranging from 1×10⁻¹²to 1×10⁻⁵ M was added for an additional 15 min. The plates werecentrifuged briefly, the incubation buffer was removed, and the cellswere lysed in lysis reagent provided with the cAMP ScintillationProximity Assay (SPA) direct screening assay system (Amersham PharmaciaBiotech Inc, Piscataway, N.J.).

[0155] The amount of cAMP (in pmol) present in the lysate was determinedfollowing instructions provided with this kit. The amount of cAMP (inpmol) produced at each concentration of peptide was plotted and analyzedby nonlinear regression using Prizm software to determine the EC₅₀ foreach peptide. The average relative potency value (ratio of the glucagonconcentration to polypeptide concentration at 50% response (EC₅₀)×100,determined from a minimum of three trials) for glucagon receptoractivation for polypeptides of the invention was <5%. The maximumactivity induced ranged from 28.3% to 71.3% of the native peptideglucagon (determined from a minimum of three trials).

[0156] The ability of the hybrid peptide to inhibit glucagon activitywas measured as follows: Following equilibration at 37° C. in a 5%CO₂/95% O₂ environment for 10 min, 10 μM peptide was added to the cellsfollowed immediately by a submaximal concentration of glucagon for 15min. The cells were lysed and cAMP determined as described above.

[0157] After subtracting the amount of cAMP produced in the unstimulatedhepatocytes from each data point, the percent inhibition was calculatedas follows: Percent inhibition is the amount of cAMP produced in thepresence of submaximal glucagon alone less the amount of cAMP producedin the presence of submaximal glucagon and 10 μM peptide, divided by theamount of cAMP produced in the presence of submaximal glucagon alone andmultiplied by 100. The average percent inhibition of glucagon activityby the polypeptides disclosed in Table 2 was between 11.3% and 59%(determined from a minimum of three trials). See Table 4.

Example 12

[0158] Measurement of Glucose Release from Rat Hepatocytes

[0159] Inhibition of glucagon-mediated glucose production as measured byglucose release from rat hepatocytes is typically at least about 20%inhibition to about 100% inhibition (i.e., 20, 30, 40, 50, 60, 70, 80,90, or 100%).

[0160] Inhibition of glucagon mediated glucose production by somepolypeptides of the invention was measured as follows. Rat hepatocyteswere added into a flat bottom 96 well plate (2×10⁵/100 μl/well) andpre-incubated in a 37° C. incubator with constant shaking and under 95%O₂/5% CO₂ flow for 10 minutes. Hepatocytes were incubated for another 30minutes after addition of glucagon with or without peptide. Cells werethen lysed with 15% perchloric acid and plates were spun at 2600 rpm, 4°C. for 15 min. The supernatant was neutralized with 1M Tris-HCl (pH8.0): 2.5 N KOH (45:55) and spun again. The resulting supernatant wasanalyzed for glucose with hexokinase and glucose-6-phosphatedehydrogenase (Methods of Enzymatic Analysis, H. U. Bermeyer, Ed.,Academic Press) and the A₃₄₀ read on a fMAX plate reader (MolecularDevices, Sunnyvale, Calif.).

[0161] Glucose output was calculated after subtracting the amount ofglucose produced in the unstimulated hepatocytes from each data pointand the percent inhibition was calculated. Percent inhibition is theamount of glucose produced in the presence of 1 nM glucagon alone lessthe amount of glucose produced in the presence of 1 nM glucagon and 100nM peptide, divided by the amount of glucose produced in the presence of1 nM glucagon alone and multiplied by 100. The polypeptide having theamino acid sequence shown in SEQ ID NO: 6 inhibited glucagon mediatedglucose production by 47% as determined from 3 trials. See Table 2.

Example 13

[0162] Measurement of Insulin Secretion by Perfused Rat Islets

[0163] Increase of insulin secretion by perfused rat islets in thisassay is an increase of at least 1.5-fold. The GLP-1 receptor agonistcomponent of polypeptides of the invention increases insulin secretionfrom perfused islets by at least 1.5-fold to about 10-fold (i.e., 1.5,2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10-fold).

[0164] Insulin secretion of perfused rat islets mediated by somepeptides of the invention was measured as follows. The bi-phasicresponses of insulin release stimulated by these polypeptides weretested by islet perfusion. Fifty islets were loaded in the perifusionchamber and perifused with HEPES-KRB containing 3 mM glucose at 37° C.After 60 min, islets were exposed to buffer containing 8 mM glucose withor without peptide (50 nM) and perifused for another 30 min. Fractionsof perifusate were collected at 1 or 5 minute intervals for insulindetermination. Insulin was measured using an ELISA kit (AlpcoDiagnostics, Windham, N.H.). At a concentration of 50 nM, thepolypeptide having the amino acid sequence shown in SEQ ID NO: 6increased insulin secretion from perfused islets approximately 2-fold,which is equivalent to that achieved by 50 μM GLP-1.

Example 14

[0165] Insulin Secretion from Dispersed Rat Islet Cells

[0166] Increase of insulin secretion from dispersed rat islet cells, inthis assay, is an increase of at least 1.5-fold. The GLP-1 receptoragonist component of such polypeptides of the invention increasesinsulin secretion from dispersed islet cells by at least 1.5-fold toabout 10-fold (i.e., 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or10-fold).

[0167] Insulin secretion of dispersed rat islets mediated by a number ofpeptides of the invention was measured as follows. Islets of Langerhanswere isolated from SD rats (200-250 g) through a digestion procedureusing collagenase. Dispersed islet cells were prepared through treatmentwith trypsin, seeded into 96 V-bottom plates and pelleted. Cells werecultured overnight in media. Media was aspirated and the cells werepre-incubated with Krebs-Ringer-HEPES buffer containing 3 mM glucose for30 minutes at 37° C. Pre-incubation buffer was removed and cells werestimulated with Krebs-Ringer-HEPES buffer containing the appropriateglucose concentration (e.g., 8 mM), with and without peptides for anappropriate time at 37° C. In some studies, an appropriate concentrationof GLP-1 also is included. A portion of supernatant was removed and itsinsulin content was measured by SPA. The results are expressed as foldover control (FOC). At a concentration of 50 nM, the polypeptide havingan amino acid sequence shown in SEQ ID NO: 27 increased insulinsecretion from dispersed islet cells approximately 3-fold. See Table 2.

Example 15

[0168] Measuring Glucagon-Stimulated Glucose Production in Fed WistarRats

[0169] Inhibition of glucagon-stimulated glucose production in thisassay is an inhibition of at least 20%. The glucagon receptor antagonistcomponent of polypeptides of the invention inhibits glucagon-mediatedelevation in blood glucose as measured by glucagon stimulated glucoseproduction in fed Wistar rats is at least 20% inhibition to about 100%inhibition (i.e., 20, 30, 40, 50, 60, 70, 80, 90, or 100%).

[0170] Glucagon stimulated glucose production in fed Wistar rats by somepolypeptides of the invention was measured as follows. Male Wistar ratswere briefly anesthetized with isoflurane gas, tail bled for bloodglucose using a Glucometer, and then given an injection into the tailvein of either vehicle (0.9% saline +1% human albumin), 0.29 nmol/kgglucagon, 1 nmol/kg polypeptides of the invention orglucagon+polypeptides of the invention. The anesthesia was withdrawn andthe conscious rats were tail-bled again after 10, 20 and 30 min.

[0171] Glucose inhibition was determined by analyzing the area under theglucose curve after subtracting the basal glucose level (dAUC). Percentinhibition is defined as the amount of glucose produced by 0.3 nmol/kgglucagon alone less the amount of glucose produced by 0.3 nmol/kgglucagon in the presence of 1 mol/kg hybrid peptide, divided by theamount of glucose produced by 0.3 nmol/kg glucagon alone and multipliedby 100.

[0172] Glucagon alone elevated blood glucose levels whereas polypeptidesof the invention alone had no effect on blood glucose levels. Thepolypeptide having the amino acid sequence shown in SEQ ID NO: 6inhibited glucagon-mediated elevation in blood glucose by 63%. See Table2.

Example 16

[0173] Measuring Glucagon-Stimulated Glucose Production in Fed Balb/CMice

[0174] Inhibition of glucose production in this assay is an inhibitionof at least about 20%. Preferably, the glucagon receptor antagonistcomponent of polypeptides of the invention inhibits glucagon-mediatedelevation in blood glucose in mice as measured by glucagon-stimulatedglucose production in fed Balb/C mice by about 20% to about 100%, (i.e.,20, 30, 40, 50, 60, 70, 80, 90, or 100%).

[0175] Glucagon stimulated glucose production in fed Balb/C mice by somepolypeptides of the invention was measured as follows. Fed male Balb/Cmice were given vehicle (0.9% saline+1% human albumin) or 100 μg/kgderivatized polypeptide by subcutaneous injection 17 hours prior to theglucagon challenge. The next day the mice were fasted for 2 hours beforereceiving an intravenous injection of 10 μg/kg glucagon or vehicle inthe tail vein. The mice were tail bled for glucose using a Glucometerjust prior to the glucagon injection and again 15 minutes afterward.

[0176] The change in glucose over the 15 minutes was calculated for eachmouse. Then the average change in glucose in the vehicle-treated groupwas subtracted from the change in glucose for each mouse in the treatedgroups to obtain the change in glucose due to glucagon. Percentinhibition is defined as the amount of glucose produced by 10 μg/kgglucagon alone less the amount of glucose produced by 10 μg/kg glucagonin the presence of 100 μg/kg hybrid peptide, divided by the amount ofglucose produced by 100 μl/kg glucagon alone and multiplied by 100. SeeTable 3. TABLE 3 Glucose (mg/dl) Delta −Vehicle % of 10 μg/kg % Group 0min 15 min (15-0) delta Glucagon Inhibition Vehicle 92 108 16 10 μg/kgglucagon 95 195 100 84 100 10 μg/kg glucagon + 75 150 75 59 69 31 100 μgpolypeptide (SEQ ID NO:27)

Example 17

[0177] Measuring Increases in Plasma Insulin Levels During In VivoGlucose Tolerance Testing (IVGTT) in Fasted Wistar Rats

[0178] An increase in plasma insulin levels in this assay is an increaseof at least about 2-fold. Preferably, the GLP-1 receptor agonistcomponent of polypeptides of the invention increases insulin secretionin rats as measured by an increase in plasma insulin levels during invivo glucose tolerance testing in fasted Wistar rats by about 2-fold toabout 5-fold, more preferably by about 2-fold to about 10-fold, andstill more preferably by about 2-fold to about 20-fold (i.e., 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20-fold).

[0179] Plasma insulin levels during in vivo glucose tolerance testing infasted Wistar rats by some polypeptides of the invention were measuredas follows. Male Wistar rats were fasted overnight and then anesthetizedwith isoflurane gas. The rats were given a tail vein injection of 0.4g/kg of glucose plus either vehicle (0.9% saline+1% albumin) or 1nmol/kg GLP-1 (positive control) or 1 nmol/kg of polypeptides of theinvention. The rats were eye-bled one minute later and the plasmaassayed for insulin using an ELISA Kit (Alpco Diagnostics (Windham,N.H.)). At a concentration of 1 nmol/kg, the polypeptide having theamino acid sequence shown in SEQ ID NO: 6 promoted insulin secretion 3-4fold, which is equivalent to that achieved by 1 nmol/kg GLP-1. See Table2.

Example 18

[0180] Effect of Peptides of the Invention on Intraperitoneal GlucoseTolerance Testing (IPGTT) in Rats or Mice

[0181] A decrease in blood glucose levels as measured by this assay is adecrease of at least about 10%. Preferably, the GLP-1 receptor agonistcomponent of polypeptides of the invention decreases blood glucoselevels in rats or mice as measured by intraperitoneal glucose tolerancetesting in rats or mice by about 10% to about 60%, more preferably byabout 10% to about 80%, still more preferably by about 10% to about 100%(i.e., 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%).

[0182] Blood glucose levels during intraperitoneal glucose tolerancetesting in rats or mice by some polypeptides of the invention weremeasured as follows. The in vivo activity of the polypeptides of theinvention when administered subcutaneously was examined in rats or mice.Rats or mice fasted overnight were given a subcutaneous injection ofcontrol or peptide (100 μg/kg). Basal blood glucose was measured priorto administration of peptides or three or 17 hours after administrationof derivatized peptides, and the rats or mice were given 2 g/kg ofglucose intraperitoneally. Blood glucose was measured again after 15, 30and 60 minutes in rats or 30 and 60 minutes in mice.

[0183] Peptides of the invention significantly reduced blood glucoselevels relative to the vehicle following the IPGTT, with 13%-54%reduction in the glucose AUC. This demonstrates that peptides haveprolonged glucose lowering activity and a prolonged half-life in vivo.GLP-1 has a very short half-life in vivo (<10 min.). The ability of thepeptides of the invention to lower blood glucose 3 hours followingpeptide administration is a clear indication that the peptide is presentin the circulation at this time point and hence has prolonged half-liferelative to GLP-1.

Example 19

[0184] Peptide PEGylation

[0185] PEGylation can be performed by any method known to those skilledin the art. However, in this instance, PEGylation was performed byintroducing a unique cysteine mutation into the peptide followed byPEGylating the cysteine via a stable thioether linkage between thesulfhydryl of the peptide and maleimide group of themethoxy-PEG-maleimide reagent (Inhale/Shearwater). It is preferable tointroduce the unique cysteine at the C-terminus of the peptide tominimize potential reduction of activity by PEGylation.

[0186] Specifically, a 2-fold molar excess of mPEG-mal (MW 22 kD and 43kD) reagent was added to 1 mg of peptide (e.g., SEQ ID NO: 25 having acysteine mutation at the C-terminus of the peptide) and dissolved inreaction buffer at pH 6 (0.1M Na phosphate/0.1M NaCl/0.1M EDTA). After0.5 hour at room temperature, the reaction was stopped with 2-fold molarexcess of DTT or free cysteine to mPEG-mal. The peptide-PEG-mal reactionmixture was applied to a cation exchange column to remove residual PEGreagents followed by gel filtration column to remove residual freepeptide. The purity, mass, and number of PEGylated sites were determinedby SDS-PAGE and MALDI-TOF mass spectrometry. PEGylation with a smallerPEG (e.g., a linear 22 kD PEG) will less likely reduce activity of thepeptide, whereas a larger PEG (e.g., a branched 43 kD PEG) will morelikely reduce activity. However, the larger PEG will increase plasmahalf life further so that once a week injection may be possible (Harris,et al., PEGylation: A Novel Process for Modifying Pharmacokinetics,Clin. Pharmacokinet 40:539-551, 2001).

Example 20

[0187] Relative Potency of GLP-1 Receptor Agonist and Glucagon ReceptorAntagonist Polypeptides

[0188] GLP-1 binding (IC₅₀ values) for polypeptides listed in Table 4was determined as described in Example 8. Relative potency values andpercent GLP-1 activity were calculated as described in Example 10. Ratsor mice were dosed with 100 μg/kg peptide and 3 hours post-dosing thedecrease in glucose levels were determined by intraperitoneal glucosetolerance testing (IPGTT) as described in Example 18. See Table 4.

[0189] Liver membrane binding (IC₅₀ values) for polypeptides listed inTable 4 were determined as described in Example 9. Percent inhibition ofglucagon stimulated cAMP increase was determined as described in Example11. Rats or mice were dosed with 100 μg/kg peptide and 17 hourspost-dosing glucose levels were determined by the methods of Examples 15and 16 and reported in Table 4. Polypeptides of the invention functionas GLP-1 receptor agonists and glucagon receptor antagonists in vivo.TABLE 4 Glucagon GLP 1 In vivo In vivo % Inhib % Decrease Decreaseglucagon glucagon RIN Relative IpGTT Liver stimulated stimulated IC50Potency % GLP1 AUC IC50 cAMP blood peptide (nM) (%) max (%)^(a) (nM)increase glucose^(b) Glucagon 1.8 GLP-1 0.4 100 100 G1 6 6.9 112.1 6.6G5 0.5 69.9 109.4 243.1 G27 2.2 13.4 98.1 69.3 31 G51 8.2 58.1 91.2154.1 35.2 G55 9.8 18.6 115.9 139.2 32.1 G56 6.6 37.2 114.8 125.3 28.6G57 14.7 18.3 132.4 177.5 18.4 G58 6.9 76.1 99.0 163.5 32.6 G59 2.1 22.7101.7 97.5 41.1 G60 13.8 22.3 95.7 682 21.8 G61 10.6 12.1 96.0 232 45.2G71 5.8 4.3 93.3 25.6 11.3 G74 4.4 8.4 88.7 21.7 23.7 G75 7.7 6.6 86.154.0 17.1 G76 10.2 7.7 85 59.1 23.0 G77 8.9 9.5 92.1 61.1 18.6 G277 ND1.8 93.7 46 65.5 G82 8.1 29.9 97.1 33.6 30.2 G83 3.2 29.9 89.4 28.6 24.7G84 1.4 61.8 113.9 11.7 25.2 G85 1.5 64.5 95.8 16.1 27.9 G185 4.5 13.0103.9 48 32 G2185 56.1 6.2 112.7 44 347 G4185 248 0.7 98.3 36 547 31 G8798.6 6.1 100.4 13 620 59 G88 109.0 2.3 118.3 16 350 G90 25.4 0.8 83.5 13726 G182 12.6 1.4 104.4 14 220 G183 158.7 0.6 90.5 27 663

[0190]

1 34 1 29 PRT Homo sapiens 1 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr SerLys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp LeuMet Asn Thr 20 25 2 30 PRT Homo sapiens 2 His Ala Glu Gly Thr Phe ThrSer Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu PheIle Ala Trp Leu Val Lys Gly Arg 20 25 30 3 31 PRT Homo sapiens 3 His AlaGlu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 GlnAla Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 4 30PRT Homo sapiens MOD_RES (30)..(30) AMIDATION 4 His Ser Gln Gly Thr PheThr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln AspPhe Val Gln Trp Leu Val Lys Gly Arg 20 25 30 5 30 PRT Homo sapiensMOD_RES (30)..(30) AMIDATION 5 His Ser Gln Gly Thr Phe Thr Ser Asp TyrSer Lys Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala TrpLeu Val Lys Gly Arg 20 25 30 6 30 PRT Homo sapiens MOD_RES (30)..(30)AMIDATION 6 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu AspAla 1 5 10 15 Arg Arg Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 2025 30 7 31 PRT Homo sapiens 7 His Ser Gln Gly Thr Phe Thr Ser Asp TyrAla Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Lys Glu Phe Ile Ala TrpLeu Val Lys Gly Arg Gly 20 25 30 8 30 PRT Homo sapiens MOD_RES(30)..(30) AMIDATION 8 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala ArgTyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Lys Glu Phe Ile Ala Trp Leu ValLys Gly Arg 20 25 30 9 30 PRT Homo sapiens MOD_RES (30)..(30) AMIDATION9 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Ala Tyr Leu Asp Ala 1 5 1015 Arg Arg Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 1030 PRT Homo sapiens MOD_RES (30)..(30) AMIDATION 10 His Ser Gln Gly ThrPhe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Ala Arg Ala LysGlu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 11 31 PRT Homo sapiens11 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 510 15 Lys Lys Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 2530 12 31 PRT Homo sapiens 12 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr AlaArg Tyr Leu Asp Ala 1 5 10 15 Lys Lys Ala Lys Glu Phe Ile Ala Trp LeuVal Lys Gly Arg Gly 20 25 30 13 31 PRT Homo sapiens 13 His Ser Gln GlyThr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Ala Lys AlaLys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 14 31 PRT Homosapiens 14 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Arg Tyr Leu AspAla 1 5 10 15 Ala Lys Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly ArgGly 20 25 30 15 31 PRT Homo sapiens 15 His Ser Gln Gly Thr Phe Thr SerAsp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Cys Glu Phe IleAla Trp Leu Val Lys Gly Arg Gly 20 25 30 16 31 PRT Homo sapiens 16 HisSer Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15Arg Arg Ala Lys Glu Phe Ile Ala Trp Leu Val Cys Gly Arg Gly 20 25 30 1731 PRT Homo sapiens 17 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala LysTyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Lys Glu Phe Ile Ala Trp Leu ValLys Cys Arg Gly 20 25 30 18 31 PRT Homo sapiens 18 His Ser Gln Gly ThrPhe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala LysGlu Phe Ile Ala Trp Leu Val Lys Gly Cys Gly 20 25 30 19 31 PRT Homosapiens 19 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu AspAla 1 5 10 15 Arg Arg Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly ArgCys 20 25 30 20 31 PRT Homo sapiens MISC_FEATURE (31)..(31) MISC_FEATURE(31)..(31) PEGylation 20 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala LysTyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Lys Glu Phe Ile Ala Trp Leu ValLys Gly Arg Cys 20 25 30 21 31 PRT Homo sapiens 21 His Ser Gln Gly ThrPhe Thr Ser Asp Tyr Ala Arg Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala LysGlu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25 30 22 31 PRT Homosapiens 22 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Arg Tyr Leu AspAla 1 5 10 15 Arg Arg Ala Arg Glu Phe Ile Lys Trp Leu Val Arg Gly ArgGly 20 25 30 23 31 PRT Homo sapiens 23 His Ser Gln Gly Thr Phe Thr SerAsp Tyr Ala Arg Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Arg Glu Phe IleAla Trp Leu Val Lys Gly Arg Gly 20 25 30 24 32 PRT Homo sapiens 24 HisSer Gln Gly Thr Phe Thr Ser Asp Tyr Ala Arg Tyr Leu Asp Ala 1 5 10 15Arg Arg Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly Lys 20 25 3025 31 PRT Homo sapiens 25 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr AlaArg Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Arg Glu Phe Ile Lys Trp LeuVal Arg Gly Arg Cys 20 25 30 26 31 PRT Homo sapiens MISC_FEATURE(31)..(31) PEGylation 26 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala ArgTyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Arg Glu Phe Ile Lys Trp Leu ValArg Gly Arg Cys 20 25 30 27 31 PRT Homo sapiens MISC_FEATURE (31)..(31)PEGylation 27 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Arg Tyr LeuAsp Ala 1 5 10 15 Arg Arg Ala Arg Glu Phe Ile Lys Trp Leu Val Arg GlyArg Cys 20 25 30 28 30 PRT Homo sapiens MOD_RES (30)..(30) AMIDATION 28His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 1015 Arg Arg Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 2931 PRT Homo sapiens LIPID (31)..(31) PALMITATE 29 His Ser Gln Gly ThrPhe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala LysGlu Phe Ile Ala Trp Leu Val Lys Gly Arg Lys 20 25 30 30 31 PRT Homosapiens LIPID (24)..(24) PALMITATE 30 His Ser Gln Gly Thr Phe Thr SerAsp Tyr Ala Arg Tyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Arg Glu Phe IleLys Trp Leu Val Arg Gly Arg Gly 20 25 30 31 32 PRT Homo sapiens LIPID(32)..(32) PALMITATE 31 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala ArgTyr Leu Asp Ala 1 5 10 15 Arg Arg Ala Arg Glu Phe Ile Lys Trp Leu ValArg Gly Arg Gly Lys 20 25 30 32 31 PRT Homo sapiens LIPID (24)..(24)PALMITATE 32 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ala Arg Tyr Leu AspAla 1 5 10 15 Arg Arg Ala Lys Glu Phe Ile Lys Trp Leu Val Arg Gly ArgGly 20 25 30 33 35 PRT Homo sapiens 33 Ile Glu Gly Arg His Ser Gln GlyThr Phe Thr Ser Asp Tyr Ala Lys 1 5 10 15 Tyr Leu Asp Ala Arg Arg AlaLys Glu Phe Ile Ala Trp Leu Val Lys 20 25 30 Gly Arg Gly 35 34 31 PRTHomo sapiens MISC_FEATURE (11)..(12) X11 = R, G, S, A, K, or T X12 = K,N, R, A, S, H, or Q 34 His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Xaa XaaTyr Leu Asp Xaa 1 5 10 15 Xaa Xaa Ala Lys Glu Phe Ile Ala Trp Leu ValLys Gly Arg Gly 20 25 30

We claim:
 1. A method of treating obesity and obesity-related disorders,comprising administering to a mammal a therapeutically effective amountof a peptide that is a GLP-1 receptor agonist and a glucagon antagonist.2. The method of claim 1, wherein the peptide is selected from the groupconsisting of SEQ ID NOs: 6 through 32, and functionally equivalentfragments, derivatives and variants thereof.
 3. The method of claim 2,wherein the peptide is selected from the group consisting of SEQ ID NO:25, SEQ ID NO: 26, and SEQ ID NO:
 27. 4. A method of treating orpreventing a disease or condition selected from the group consisting ofdiabetes (Type 2), maturity-onset diabetes of the young (MODY), latentautoimmune diabetes adult (LADA), impaired glucose tolerance (IGT),impaired fasting glucose (IFG), gestational diabetes, and metabolicsyndrome X, comprising administering to a mammal an effective amount ofa peptide that is a GLP-1 receptor agonist and a glucagon antagonist. 5.The method of claim 4, wherein the peptide is selected from the groupconsisting of SEQ ID NOs: 6 through 32, and functionally equivalentfragments, derivatives and variants thereof.
 6. The method of claim 5,wherein the peptide is selected from the group consisting of SEQ ID NO:25, SEQ ID NO: 26, and SEQ ID NO:
 27. 7. The method of claim 4, furthercomprising administering a PPAR-agonist, an insulin sensitizer, asulfonylurea, an insulin secretagogue, a hepatic glucose output loweringcompound, an α-glucosidase inhibitor or insulin in combination with saidpeptide.
 8. The method of claim 7, wherein said PPAR-agonist is selectedfrom rosiglitazone and pioglitazone.
 9. The method of claim 7, whereinsaid sulfonylurea is selected from glibenclamide, glimepiride,chlorpropamide, and glipizide.
 10. The method of claim 7, wherein saidinsulin secretagogue is selected from GLP-1, GIP, PAC/VPAC receptoragonists, secretin, nateglinide, meglitinide, repaglinide,glibenclamide, glimepiride, chlorpropamide, and glipizide.
 11. Themethod of claim 7, wherein said α-glucosidase inhibitor is selected fromacarbose, miglitol and voglibose.
 12. The method of claim 7, whereinsaid hepatic glucose output lowering compound is metformin.
 13. Themethod of claim 4, further comprising administering an HMG-CoA reductaseinhibitor, nicotinic acid, a bile acid sequestrant, a fibric acidderivative, antihypertensive drug, or an anti-obesity drug incombination with said peptide.
 14. The method of claim 13, wherein saidanti-obesity drug is selected from a β-3 agonist, a CB-1 antagonist, anda lipase inhibitor.
 15. A method of treating or preventing secondarycauses of diabetes selected from glucocorticoid excess, growth hormoneexcess, pheochromocytoma, and drug-induced diabetes, comprisingadministering to a mammal an effective amount of a a peptide that is aGLP-1 receptor agonist and a glucagon antagonist.
 16. The method ofclaim 15, wherein the peptide is selected from the group consisting ofSEQ ID NOs: 6 through 32, and functionally equivalent fragments,derivatives and variants thereof.
 17. The method of claim 16, whereinthe peptide is selected from the group consisting of SEQ ID NO: 25, SEQID NO: 26, and SEQ ID NO:
 27. 18. A method of increasing the sensitivityof pancreatic beta cells to an insulin secretagogue, comprisingadministering to a mammal an effective amount of a a peptide that is aGLP-1 receptor agonist and a glucagon antagonist.
 19. The method ofclaim 18, wherein the peptide is selected from the group consisting ofSEQ ID NOs: 6 through 32, and functionally equivalent fragments,derivatives and variants thereof.
 20. The method of claim 19, whereinthe peptide is selected from the group consisting of SEQ ID NO: 25, SEQID NO: 26, and SEQ ID NO:
 27. 21. The method of claim 18, wherein saidinsulin secretagogue is selected from GLP-1, GIP, PAC/VPAC receptoragonists, secretin, nateglinide, meglitinide, repaglinide,glibenclamide, glimepiride, chlorpropamide, and glipizide.